Immunization with vaccinia virus resulted in long-lasting protection against smallpox and was the approach used to eliminate natural smallpox infections worldwide. This was accomplished without a detailed understanding of human T cell responses to poxviruses. Due to concerns about the use of smallpox virus as a bioweapon, smallpox vaccination is currently being reintroduced. Considering the relatively high incidence of side effects, developing a safer, but effective vaccine is very important. Vaccinia virus elicits strong cellular as well as humoral immune responses. Cellular immunity seems to be more important for recovery from infection. Severe complications from vaccination were associated with congenital or acquired T cell deficiencies, but not with congenital agammaglobulinemia. The presence of neutralizing antibody alone did not prevent the development of progressive vaccinia if cell-mediated immunity was defective. In order to analyze human T cell responses to licensed and experimental smallpox vaccines at the single cell level, it is essential to identify T cell epitopes, especially immunodominant CD8 + T cell epitopes. Vaccinia is a large virus and we hypothesize that we can develop a rapid approach to localize gene fragments encoding human T cell epitopes and to identify them precisely using peptides using PCR-generated DNA fragments containing virus genes transfected into antigen presenting cells. Complementary strategies will employ peptide libraries and studies in transgenic mice expressing common human MHC class I molecules. The immunodominance of human T cell epitopes on vaccinia virus will be analyzed. The results of this research project will provide valuable information relative to basic studies of human T cell memory and data useful for the design and analyses of experimental smallpox vaccines. The methods we will establish in this project may be applicable to other large viruses as well as bacteria for the definition of human T cell epitopes.